Microorganisms such as bacterial pathogens can be difficult to cultivate from complex clinical and environmental samples. They may be present in small numbers or in injured and aged physiological states with poor plating efficiency. Samples often have competing microbial flora that overgrow pathogens on non-selective media, while selective media can reduce yield and select against some strains. Most culture-based detection methods require 1-3 days to yield results, too slow for many circumstances, especially life-threatening ones.
An alternative to bacteriological culture is nucleic acid amplification testing (NAAT). The most common type of NAAT, the polymerase chain reaction (PCR), is rapid and sensitive. A limitation of PCR is its inability to distinguish viable pathogen cells from non-viable cells, from free nucleic acids in samples, and from contaminating nucleic acids introduced during the testing process. PCR is also mechanistically complex and susceptible to inhibition by substances in samples. These limitations are especially problematic when PCR is used to assess the efficacy of antimicrobial treatment, disinfection (e.g. water treatment), and clean-up processes.
In order to improve the sensitivity and specificity of NAAT for viable microorganisms it would be valuable to reduce or eliminate the false-positive detection of non-viable microorganisms and free DNA. One approach is the detection of microbial RNA rather than DNA. RNA is considered less stable than DNA in solution and in dead cells. Species-specific probes for ribosomal RNA (rRNA) or messenger RNA (mRNA) are known. However, microbial mRNA is difficult to detect due to its instability and low abundance (Gedalanga and Olson. 2009. Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples. Appl Microbiol Biotechnol. 82:587-596). Conversely, mature rRNA is fairly stable and can persist within dead bacterial cells for long periods of time.